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Determination of Survival of Wildtype and Mutant Escherichia coli in Soil
野生型和突变型大肠埃希杆菌在土壤中的存活测定   

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Abstract

E. coli resides in the gastrointestinal tract of humans and other warm-blooded animals but recent studies have shown that E. coli can persist and grow in various external environments including soil. The general stress response regulator, RpoS, helps E. coli overcome various stresses, however its role in soil survival was unknown. This soil survival assay protocol was developed and used to determine the role of the general stress response regulator, RpoS, in the survival of E. coli in soil. Using this soil survival assay, we demonstrated that RpoS was important for the survival of E. coli in soil. This protocol describes the development of the soil survival assay especially the recovery of E. coli inoculated into soil and can be adapted to allow further investigations into the survival of other bacteria in soil.

Keywords: Soil survival(土壤生存), Escherichia coli(大肠埃希杆菌), RpoS(RpoS), Environmental persistence(环境持久性), Bacteria recovery(细菌恢复)

Background

Escherichia coli is a Gram-negative, facultative anaerobe, belonging to the Enterobacteriaceae family, which inhabits the intestinal tract of humans, warm-blooded animals and reptiles (Berg, 1996; Gordon and Cowling, 2003). It can be transferred through water and sediments via faeces and is used as an indicator of faecal contamination in drinking and recreational water. The use of E. coli as a faecal indicator is based, at least in part, on the assumption that it exists transiently outside of the host gastrointestinal tract (Ishii and Sadowsky, 2008) and does not survive for a long time in the external environment. However, several studies have isolated E. coli from various natural environments such as municipal wastewater, freshwater, beach water, beach sand and soils (Jiménez et al., 1989; Brennan et al., 2010; Chiang et al., 2011; Byappanahalli et al., 2012; Zhi et al., 2016). The capacity of these E. coli strains to survive for long periods of time and grow in the external environment raises questions about the validity of its continued use as indicator of water quality (Brennan et al., 2010). To understand the genetic mechanism underlying the survival and persistence of E. coli in soil, we developed a soil survival assay to evaluate the role of the different genetic factors on soil survival. We investigated the role of the general stress response regulator, RpoS, in the survival of long-term soil persistent E. coli in soil. The ability of the rpoS mutant (COB583ΔrpoS) to survive in soil was compared with the wildtype strain (COB583) and RpoS was demonstrated to be important for the survival and long-term persistence of E. coli in soil (Somorin et al., 2016). Here, we present the detailed protocol for the soil survival assay and the recovery of E. coli from soil.

Materials and Reagents

Note: All reagents used were from the specified manufacturers (catalog numbers indicated). Nonetheless, the same reagents from different manufacturers are expected to produce similar results.

  1. Spatula
  2. Weighing boat (Sparks Lab Supplies, catalog number: BAL1822 )
  3. Gloves (Sparks Lab Supplies, catalog number: SAF5534X20 )
  4. Sterile micropipette tips (100-1,000 µl) (Abdos Labtech, catalog number: P10102 )
  5. Sterile micropipette tips (2-200 µl) (Abdos Labtech, catalog number: P10101 )
  6. Microcentrifuge tube (1.5 ml) (Abdos Labtech, catalog number: P10202 )
  7. Centrifuge tube (15 ml graduated) (Abdos Labtech, catalog number: P10402 )
  8. Cuvette (LP ITALIANA, catalog number: 112117 )
  9. 2 mm laboratory test sieve (B5410/1986, Endecotts, catalog number: 200SIW2.00 )
  10. 96-well plate (TC microwell 96U) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 163320 )
  11. 90 mm Petri dishes (Abdos Labtech, catalog number: P10901 )
  12. Silty-Loam soil
  13. E. coli strains
    1. E. coli COB583 (Somorin et al., 2016)
    2. E. coli COB583ΔrpoS (Somorin et al., 2016)
    Note: E. coli stock cultures were made in LB with 7% Dimethyl sulfoxide (v/v; Sigma-Aldrich) and kept at -80 °C pending use.
  14. Luria-Bertani (LB) broth (Sigma-Aldrich, catalog number: L3022-1KG )
  15. Agar No. 2 (Lab M, catalog number: MC006 )
  16. MacConkey agar (Sigma-Aldrich, catalog number: M7408 )
  17. Distilled water (Milli-Q) (EMD Millipore)
  18. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417-100TAB )
  19. LB broth (see Recipes)
  20. LB agar plates (see Recipes)
  21. PBS buffer (see Recipes)
  22. MacConkey agar plates (see Recipes)

Equipment

  1. 250 ml conical flask (VWR, catalog number: 214-1132 )
  2. -80 °C freezer
  3. Styrofoam 15 ml tube holder
  4. Bunsen burner
  5. Orbital shaker (Gallenkamp)
  6. Discovery comfort multichannel pipette (20-200 µl; HTL)
  7. Discovery comfort multichannel pipette (5-20 µl; HTL)
  8. Nichipet EX pipette (200-1,000 µl; Nichiryo)
  9. Nichipet EX Pipetman classic pipettes (20-200 µl; Nichiryo)
  10. Vortex mixer (Reax top; Heidolph)
  11. Biomate 3 spectrophotometer (Thermo Fisher Scientific, catalog number: 335904 )
    Note: This product has been discontinued.
  12. Weighing scale (Sartorius, catalog number: BL120S )
    Note: This product has been discontinued.
  13. Centrifuge (Eppendorf, model: 5418 )
  14. Labo autoclave (SANYO, model: MLS-3020U )

Procedure

  1. Measurement of cell concentration by optical density (OD600 nm)
    1. Streak out Escherichia coli COB583 wildtype and the rpoS deletion mutant (COB583ΔrpoS) from the stock culture kept in the -80 °C freezer onto LB agar (see Recipes) around a Bunsen burner and incubate them overnight (~16 h) at 37 °C.
    2. Inoculate one colony of each strain with a disposable sterile inoculating loop into 10 ml LB in a sterile conical flask around a Bunsen burner and incubate them at 37 °C for 6 h. Perform this step in triplicate for each strain.
    3. From the 6 h culture, take 100 µl and add it to 900 µl LB and determine the OD600 nm.
    4. Determine the volume of the 6 h culture required to give a starting OD600 nm of 0.05 in a 25 ml LB as follows:
      C1V1 = C2V2
      C1 = OD600 nm of the 6 h culture; V1 = volume of the 6 h culture
      C2 = final OD600 nm required; V2 = final volume required
      V1 = 0.05 x 25 ml/C1
    5. Make the cultures with starting OD600 nm of 0.05 in 25 ml LB for each strain and their respective replicates in sterile 250 ml conical flasks and incubate the flasks overnight on a shaker at 37 °C.
    6. On the following day, dilute the overnight cultures 1:10 in LB and determine the OD600 nm.
    7. Pipette 1 ml of each overnight culture into two separate sterile 1.5 ml tubes for each strain and their respective replicates.
    8. Centrifuge the cultures at 9,000 x g for 10 min at room temperature (23-25 °C) and remove the supernatants with sterile pipette.
    9. Wash the cell pellets by re-suspending them in sterile PBS (see Recipes) and centrifuge at 9,000 x g for 10 min. Repeat the washing step two more times.
    10. Resuspend the washed cell pellets in 1 ml of sterile PBS by pipetting up and down and vortexing and dilute 1:2; 1:5; 1:10; 1:20; 1:100; and 1:1,000 in sterile PBS to make 1 ml in sterile microcentrifuge tubes.
    11. Transfer each diluted cell suspension into a cuvette and determine the OD600 nm of the diluted cell suspensions of the E. coli strains and their respective replicates in a spectrophotometer three times and determine the mean value.
    12. Make serial dilutions of each cell suspension and spot 10 µl onto LB plates in triplicate and incubate the plates at 37 °C overnight.
    13. Count the colonies and use the numbers derived to estimate the cell count (CFU ml-1) and determine the mean cell count from the triplicate experiments.
    14. Plot the mean cell count against the mean OD600 nm of the respective dilutions of each strain in three independent experiments to obtain calibration curves from which desired cell numbers can be obtained. The standard curve of graph of the cell count versus OD600 nm is presented in Figure 1.


      Figure 1. Standard curve of cell numbers versus OD600 nm of COB583 (A) and COB583ΔrpoS (B)

  2. Development of soil survival assay
    1. Inoculate one colony each of COB583 and COB583ΔrpoS into 25 ml LB in triplicate and incubate overnight at 37 °C.
    2. Dilute the resulting cultures 1:10 in LB and determine the OD600 nm.
    3. Add 1 ml of the overnight culture to two sterile Eppendorf tubes for each replicate of the two strains.
    4. Wash the cultures as described above (steps A8 and A9).
    5. Pool the resulting cell pellets and resuspend in 2 ml of sterile PBS in a 15 ml tube.
    6. Make 1:10 dilution (1 ml) of the each washed cell suspension to determine the OD600 nm.
    7. Based on the OD600 nm, prepare a normalised cell suspension with OD600 nm equivalent to 2 x 108 CFU ml-1 from each replicate.
    8. Make serial dilution of each normalised bacterial suspension and spot 10 µl of each dilution on LB agar in triplicate. Allow the spots to air-dry and incubate the plates overnight at 37 °C. Count the colonies and determine the mean cell counts.
    9. Sieve the soil to be used with 2 mm laboratory test sieve and weigh 1 g of the sieved soil into a 15 ml tube.
    10. Take 50 µl of each normalised bacterial suspension for each strain and inoculate it into the soil (to give ~1 x 107 CFU g-1 of soil). The uninoculated control should have 50 µl of sterile PBS added to it. The experiment was set up in triplicate for each strain.
    11. Cover the tubes with the lid and invert them 10 times by hand for proper mixing.
    12. Leave the tubes for about 10 min at room temperature (23-25 °C).
    13. Perform the recovery of the inoculated E. coli strains by adding 2 ml of sterile PBS to each 15 ml tube. Cover the tube with its lid, invert it three times and immediately vortex for 20 sec twice.
    14. Leave the tube on the workbench and allow the larger soil particles in the resulting soil slurry to settle out (2-10 min, depending on soil texture) (Figure 2).


      Figure 2. Experimental set-up for the soil survival assay with the soil slurry just after mixing and after settling out

    15. Take 20 µl from the supernatant of each soil slurry, place them in separate wells in a 96-well plate and make serial dilution (up to 10-6) from them.
    16. Take 10 µl of each dilution and spot onto MacConkey agar plates (see Recipes) in triplicate, and incubate plates overnight at 37 °C.
    17. Count the colonies and use the numbers derived to estimate the mean recovered cell count (CFU ml-1) from the triplicate experiments. The percentage recovery of cells was determined. 

Data analysis

Cell count for each replicate is represented by the mean cell counts obtained from the three spots in each replicate. The mean and standard deviation of the cell counts from the normalised cell suspension (inoculum) and recovered cell counts were obtained from three independent replicates of each strain. The percentage of recovery ranges from 30-79% and the recovered cells from this procedure is presented in Figure 3.




Figure 3. Recovery of Escherichia coli from soil. Percentage recovery is provided above the recovered cells.

Notes

  1. The optical density of the inoculum should be confirmed to be accurate before inoculating into soil.
  2. Care should be taken when inoculating the soil to ensure the pipette tips do not contact the soil directly, as it prevents the release of the entire content of the pipette tip.
  3. Care should be taken when spotting the cell suspension on agar plates so as not to puncture the agar with the pipette tips. This could vary the cell numbers arising from each spot.
  4. The soil to be used should be checked and confirmed not to contain E. coli prior to the soil survival assay so that recovered E. coli will only be attributable to inoculated E. coli. Serial dilutions of 1 g of the soil sieved through a 2 mm mesh should be plated on MacConkey agar and incubated at 37 °C to determine this.
  5. For long-term soil survival experiments, the tubes should be slightly capped to allow air exchange and incubated statically at the desired temperature. Multiple tubes should be set up for experiments requiring sampling at different time points since inoculated soils are destructively sampled (i.e., PBS was added and could not be reused at another time point) for E. coli recovery.
  6. No significant difference was seen when soil slurry was left to settle for 2 and 10 min before aliquots were taken for serial dilution and plating for cell count determination.

Recipes

  1. LB broth
    Add 20 g LB powder to 1 L of distilled water and autoclave
  2. LB agar plates
    Add 20 g LB powder and 15 g agar No. 2 powder to 1 L of distilled water and autoclave. When cool to touch (~ 45 °C), pour about 25 ml of the molten agar into Petri dishes and allow to solidify
  3. PBS buffer
    Dissolve one phosphate-buffered saline tablet (Sigma-Aldrich) in 200 ml of distilled water and autoclave, giving a PBS buffer (pH 7.4)
  4. MacConkey agar plates
    Add 50 g MacConkey agar powder to 1 L of distilled water and autoclave. When cool to touch (~45 °C), pour about 25 ml of the molten agar into Petri dishes and allow to solidify

Acknowledgments

The protocol was adapted from a previously published work (Somorin et al., 2016). The authors thank Camila Thorne for her help and advice with setting up the soil survival assay. This work was supported by an NUI Galway College of Science Ph.D. Fellowship and Thomas Crawford Hayes research award to Y.S.

References

  1. Anderson, K. L., Whitlock, J. E. and Harwood, V. J. (2005). Persistence and differential survival of fecal indicator bacteria in subtropical waters and sediments. Appl Environ Microbiol 71(6): 3041-3048.
  2. Berg, R. D. (1996). The indigenous gastrointestinal microflora. Trends Microbiol 4(11): 430-435.
  3. Brennan, F. P., O'Flaherty, V., Kramers, G., Grant, J. and Richards, K. G. (2010). Long-term persistence and leaching of Escherichia coli in temperate maritime soils. Appl Environ Microbiol 76(5): 1449-1455.
  4. Byappanahalli, M. N., Whitman, R. L., Shively, D. A., Sadowsky, M. J. and Ishii, S. (2006). Population structure, persistence, and seasonality of autochthonous Escherichia coli in temperate, coastal forest soil from a Great Lakes watershed. Environ Microbiol 8(3): 504-513.
  5. Byappanahalli, M. N., Yan, T., Hamilton, M. J., Ishii, S., Fujioka, R. S., Whitman, R. L. and Sadowsky, M. J. (2012). The population structure of Escherichia coli isolated from subtropical and temperate soils. Sci Total Environ 417-418: 273-279.
  6. Chiang, S. M., Dong, T., Edge, T. A. and Schellhorn, H. E. (2011). Phenotypic diversity caused by differential RpoS activity among environmental Escherichia coli isolates. Appl Environ Microbiol 77(22): 7915-7923.
  7. Gordon, D. M. and Cowling, A. (2003). The distribution and genetic structure of Escherichia coli in Australian vertebrates: host and geographic effects. Microbiology 149(Pt 12): 3575-3586.
  8. Jimenez, L., Muniz, I., Toranzos, G. A. and Hazen, T. C. (1989). Survival and activity of Salmonella typhimurium and Escherichia coli in tropical freshwater. J Appl Bacteriol 67(1): 61-69.
  9. Ishii, S., Ksoll, W. B., Hicks, R. E. and Sadowsky, M. J. (2006). Presence and growth of naturalized Escherichia coli in temperate soils from Lake Superior watersheds. Appl Environ Microbiol 72(1): 612-621.
  10. Ishii, S. and Sadowsky, M. J. (2008). Escherichia coli in the environment: Implications for water quality and human health. Microbes Environ 23(2): 101-108.
  11. Somorin, Y., Abram, F., Brennan, F. and O'Byrne, C. (2016). The general stress response is conserved in long-term soil-persistent strains of Escherichia coli. Appl Environ Microbiol 82(15): 4628-4640.
  12. Zhi, S., Banting, G., Li, Q., Edge, T. A., Topp, E., Sokurenko, M., Scott, C., Braithwaite, S., Ruecker, N. J., Yasui, Y., McAllister, T., Chui, L. and Neumann, N. F. (2016). Evidence of naturalized stress-tolerant strains of Escherichia coli in municipal wastewater treatment plants. Appl Environ Microbiol 82(18): 5505-5518.

简介

电子。 大肠杆菌位于人类和其他温血动物的胃肠道中,但最近的研究表明,E。 大肠杆菌可以在包括土壤在内的各种外部环境中持续生长。 一般的压力响应调节器RpoS有助于E。 克隆各种压力,但其在土壤中的作用尚不清楚。 开发了这种土壤生存测定方案,并用于确定一般应激反应调节剂RpoS在E的存活中的作用。 大肠杆菌在土壤中。 使用这种土壤生存测定,我们证明RpoS对于E的生存是重要的。 大肠杆菌在土壤中。 该方案描述了土壤生存测定的发展,特别是E的恢复。 大肠杆菌接种到土壤中,可适应于进一步研究土壤中其他细菌的存活。
【背景】大肠杆菌是属于肠杆菌科的革兰氏阴性兼性厌氧菌,其栖息于人类的肠道,温血动物和爬行动物(Berg,1996; Gordon和Cowling,2003)。可通过粪便通过水和沉淀物进行转运,并用作饮用水和娱乐用水中粪便污染的指标。使用 E。大肠杆菌作为粪便指标至少部分地基于其临时存在于主体胃肠道之外的假设(Ishii和Sadowsky,2008),并且在外部环境中长时间不存活。然而,一些研究已经分离出E。来自各种自然环境的大肠杆菌,例如城市废水,淡水,沙滩水,海滩沙土和土壤(Jiménez等人,1989; Brennan等人, 2010; Chiang等人,2011; Byappanahalli等人,2012; Zhi等人,2016)。这些E的能力。大肠杆菌菌株长时间存活并在外部环境中生长提出了关于其继续使用作为水质指标的有效性的问题(Brennan等人,2010)。了解E的生存和持久性的遗传机制。大肠杆菌在土壤中,我们开发了土壤生存测定,以评估不同遗传因素对土壤存活的作用。我们调查了一般应激反应调节剂RpoS在长期土壤持续性E的存活中的作用。大肠杆菌在土壤中。将土壤中的rpoS突变体(COB583Δ)在野生型中的能力与野生型菌株(COB583)进行比较,并证明RpoS对于存活和长期存活是重要的,长期坚持E。土壤中的大肠杆菌(Somorin等人,2016)。在这里,我们介绍了土壤生存测定和E恢复的详细方案。大肠杆菌从土壤。

关键字:土壤生存, 大肠埃希杆菌, RpoS, 环境持久性, 细菌恢复

材料和试剂

注意:所有使用的试剂均来自指定的制造商(目录号)。尽管如此,来自不同制造商的相同试剂也有望产生类似的结果

  1. Spatula
  2. 称重船(Sparks Lab Supplies,目录号:BAL1822)
  3. 手套(Sparks Lab Supplies,目录号:SAF5534X20)
  4. 无菌微量吸头(100-1,000μl)(Abdos Labtech,目录号:P10102)
  5. 无菌微量吸头(2-200μl)(Abdos Labtech,目录号:P10101)
  6. 微量离心管(1.5ml)(Abdos Labtech,目录号:P10202)
  7. 离心管(15 ml刻度)(Abdos Labtech,目录号:P10402)
  8. Cuvette(LP ITALIANA,目录号:112117)
  9. 2 mm实验室试验筛(B5410 / 1986,Endecotts,目录号:200SIW2.00)
  10. 96孔板(TC microwell 96U)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:163320)
  11. 90毫米培养皿(Abdos Labtech,目录号:P10901)
  12. 土壤土壤
  13. 大肠杆菌菌株
    1. 电子。大肠杆菌COB583(Somorin等人,2016)
    2. 电子。大肠杆菌COB583Δ(Somorin等人,2016)
    注意:大肠杆菌储存培养物在含有7%二甲基亚砜(v / v; Sigma-Aldrich)的LB中制备,并保持在-80℃待用。
  14. Luria-Bertani(LB)肉汤(Sigma-Aldrich,目录号:L3022-1KG)
  15. 琼脂2号(实验室M,目录号:MC006)
  16. MacConkey琼脂(Sigma-Aldrich,目录号:M7408)
  17. 蒸馏水(Milli-Q)(EMD Millipore)
  18. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P4417-100TAB)
  19. LB肉汤(见食谱)
  20. LB琼脂平板(参见食谱)
  21. PBS缓冲液(参见食谱)
  22. MacConkey琼脂平板(参见食谱)

设备

  1. 250毫升锥形瓶(VWR,目录号:214-1132)
  2. -80°C冰箱
  3. 泡沫塑料15毫升管夹
  4. 本生灯
  5. 轨道摇床(Gallenkamp)
  6. 发现舒适多通道移液器(20-200μl; HTL)
  7. 发现舒适多通道移液管(5-20μl; HTL)
  8. Nichipet EX吸管(200-1,000μl; Nichiryo)
  9. Nichipet EX Pipetman经典移液器(20-200μl; Nichiryo)
  10. 涡旋搅拌机(Reax top; Heidolph)
  11. Biomate 3分光光度计(Thermo Fisher Scientific,目录号:335904)
    注意:本产品已停产。
  12. 称重秤(Sartorius,目录号:BL120S)
    注意:本产品已停产。
  13. 离心机(Eppendorf,型号:5418)
  14. Labo高压釜(SANYO,型号:MLS-3020U)

程序

  1. 通过光密度(OD 600nm)测量细胞浓度
    1. 将保存在-80℃冷冻箱中的储存培养物中的大肠杆菌COB583野生型和缺失型突变体(COB583ΔnPoS)分离到LB琼脂(参见食谱)在本生灯周围,并在37℃孵育过夜(〜16小时)。
    2. 将一个菌株的一个菌落用一次性无菌接种环接种在本氏燃烧器周围的无菌锥形瓶中的10ml LB中,并在37℃孵育6小时。对每个菌株进行一式三份的这一步骤。
    3. 从6小时培养物中取100μl加入到900μlLB中并测定OD 600nm
    4. 确定在25ml LB中使起始OD 600nm <0.05的所需培养物的体积如下:
      C 1 1 2 2&lt; 2&gt;
      6小时培养物的C = 600nm; V <1> = 6小时培养物的体积 C 2 O 2 =所需的最终OD 600nm ; V 2 =最终卷需要
      V 1 = 0.05×25ml /℃1 / / 2
    5. 在每个菌株的25ml LB中制备起始OD 600nm <0.05的培养物,并将它们各自在无菌250ml锥形瓶中重复,并在37℃下在振荡器上温育烧瓶过夜。 >
    6. 第二天,在LB中稀释1:10的过夜培养物,并测定OD 600nm
    7. 将1ml每个过夜培养物移取到每个菌株的两个分开的无菌的1.5ml管中,并分别进行复制
    8. 在室温(23-25℃)下以9,000 x g离心培养物10分钟,用无菌移液管除去上清液。
    9. 通过将其悬浮在无菌PBS(参见食谱)中并以9,000×g离心10分钟来洗涤细胞沉淀。重复洗涤步骤两次。
    10. 通过上下吸取将洗涤的细胞沉淀物重悬于1ml无菌PBS中并涡旋并稀释1:2; 1:5; 1:10; 1:20; 1:100;和1:1,000在无菌PBS中,使其在无菌微量离心管中制成1ml
    11. 将每个稀释的细胞悬浮液转移到比色皿中,并确定E细胞悬液的OD 600nm。大肠杆菌菌株及其各自的重复在分光光度计中三次并确定平均值。
    12. 对每个细胞悬浮液进行连续稀释,并将其放在LB平板上一式三份,并在37℃孵育平板过夜。
    13. 计数菌落,并使用派生的数据估计细胞计数(CFU ml -1 ),并从一式三份的实验中确定平均细胞数。
    14. 在三个独立实验中绘制平均细胞数相对于每个菌株的相应稀释度的平均OD 600nm,以获得可从其获得所需细胞数的校准曲线。细胞计数与OD 600 nm 的曲线图的标准曲线如图1所示

      图1. COB583(A)和COB583ΔrpoS的细胞数与OD 600的相对标准曲线(B)

  2. 土壤生存测定的发展
    1. 将COB583和COB583ΔrpoS各自的一个菌落一式三份接种到25ml LB中,并在37℃下孵育过夜。
    2. 在LB中稀释所得培养物1:10并测定OD 600nm
    3. 将2ml过夜培养物加入到两个无菌Eppendorf管中,每个重复两个菌株。
    4. 如上所述洗涤培养物(步骤A8和A9)。
    5. 将所得细胞沉淀物收集并重悬于2ml无菌PBS中的15ml试管中
    6. 将每个洗涤的细胞悬浮液稀释1:10(1ml),以确定OD 600nm
    7. 基于OD600nm,从每个重复制备具有OD 600纳米相当于2×10 8 CFU ml -1的标准化细胞悬浮液。
    8. 对每个标准化的细菌悬浮液进行连续稀释,并在LB琼脂上点样10μl每种稀释液一式三份。让斑点风干,并在37℃孵育板过夜。计数菌落并确定平均细胞计数。
    9. 用2毫米实验室试验筛筛分土壤,称取1克筛分土壤至15毫升管中。
    10. 取每个菌株每个标准化细菌悬浮液50μl,并将其接种到土壤中(给予〜1×10 7个土壤的CFU g 1)。未接种的对照物应该加入50μl无菌PBS。实验为每个菌株设置一式三份。
    11. 用盖子盖住管子,用手将它们倒置10次以进行适当的混合。
    12. 在室温(23-25°C)下放置管约10分钟。
    13. 执行接种的E的恢复。通过向每个15ml管中加入2ml无菌PBS至大肠杆菌菌株。用盖盖住管子,反复三次,立即旋转20秒两次。
    14. 将管放在工作台上,使得所得土壤泥浆中较大的土壤颗粒沉降(2-10分钟,取决于土壤质地)(图2)。


      图2.刚刚混合后和沉淀后的土壤泥浆土壤生存测定的实验设置

    15. 从每个土壤浆液的上清液中取20μl,将它们置于96孔板的分开的孔中,并从其中进行连续稀释(最多10次至-6℃)。
    16. 取10μl每种稀释液,并一式三份放在MacConkey琼脂平板上(见食谱),并在37℃下孵育板过夜。
    17. 计算菌落,并使用得到的数据从一式三份的实验中估计平均回收细胞数(CFU ml -1 )。确定细胞的回收百分比。&nbsp;

数据分析

每个重复的细胞计数由从每个重复中的三个点获得的平均细胞计数表示。从标准化的细胞悬浮液(接种物)中获得细胞计数的平均值和标准偏差,并从每个菌株的三个独立重复获得回收的细胞计数。恢复的百分比范围为30-79%,来自该程序的回收细胞如图3所示



图3.从土壤中回收大肠杆菌 在回收细胞上提供百分比恢复。

笔记

  1. 在接种土壤前,应确认接种物的光密度准确
  2. 接种土壤时应注意确保移液器吸头不要直接接触土壤,因为它可以防止移液器吸头的整个内容物的释放。
  3. 当细胞悬浮液在琼脂平板上发现时,应注意使用移液器吸头刺穿琼脂。这可能会改变每个地点产生的细胞数。
  4. 应检查使用的土壤,并确认不含有E。在土壤存活测定之前,使E.coli 恢复。大肠杆菌只能归因于接种的E.大肠杆菌。将通过2mm筛网筛选的1g土壤的系列稀释液涂布在MacConkey琼脂上,并在37℃下孵育以确定其结果。
  5. 对于长期的土壤生存实验,管应该稍微加盖以允许空气交换并在所需温度下静态温育。应设置多个试管,用于在不同时间点进行抽样的实验,因为接种的土壤被破坏性采样(即添加了PBS,不能在另一个时间点重新使用)。大肠杆菌恢复。
  6. 当等分试样连续稀释和电镀细胞计数确定之前,将土壤浆液静置2分钟和10分钟后,没有显着差异。

食谱

  1. LB肉汤
    向1升蒸馏水中加入20克LB粉末,并加热高压釜
  2. LB琼脂板
    向1L蒸馏水中加入20g LB粉末和15g琼脂2粉末,并加热。当冷却触摸(约45°C)时,将约25ml的熔融琼脂倒入培养皿中,并使其固化。
  3. PBS缓冲液
    将一个磷酸盐缓冲盐水溶液(Sigma-Aldrich)溶解在200ml蒸馏水中并进行高压灭菌,得到PBS缓冲液(pH 7.4)
  4. MacConkey琼脂板
    将50克MacConkey琼脂粉末加入1升蒸馏水中并加热。当冷却触摸(约45°C)时,将约25ml的熔融琼脂倒入培养皿中,并使其固化。

致谢

该协议是从以前发表的作品(Somorin等人,2016)改编而成。作者感谢Camila Thorne的帮助和建议,建立了土壤生存测定。这项工作由NUI戈尔韦理工学院博士支持。奖学金和Thomas Crawford Hayes研究奖给Y.S.

参考

  1. Anderson,KL,Whitlock,JE and Harwood,VJ(2005)。&nbsp; 亚热带水域和沉积物中粪便指示剂细菌的持续存在和差异生存。应用环境微生物71(6):3041-3048。
  2. Berg,RD(1996)。土着胃肠道菌群。 趋势微生物 4(11):430-435。
  3. Brennan,FP,O'Flaherty,V.,Kramers,G.,Grant,J.and Richards,KG(2010)。&lt; a class =“ke-insertfile”href =“http://www.ncbi。 nlm.nih.gov/pubmed/20038692“target =”_ blank“>温带海洋土壤中大肠埃希菌的长期持续和浸出。 Appl Environ Microbiol 76(5):1449-1455。
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  5. Byappanahalli,MN,Yan,T.,Hamilton,MJ,Ishii,S.,Fujioka,RS,Whitman,RL和Sadowsky,MJ(2012)。&lt; a class =“ke-insertfile”href =“http: /www.ncbi.nlm.nih.gov/pubmed/22264918“target =”_ blank“>从亚热带和温带土壤分离的大肠埃希菌的种群结构。科学总量Environ 417-418:273-279。
  6. Chiang,SM,Dong,T.,Edge,TA and Schellhorn,HE(2011)。&nbsp; 环境大肠杆菌分离株中不同RpoS活性引起的表型多样性 Appl Environ Microbiol 77 /(22):7915-7923 。
  7. Gordon,DM and Cowling,A.(2003)。&nbsp; 澳大利亚脊椎动物中大肠埃希氏菌的分布和遗传结构:宿主和地理效应。 149(Pt 12):3575-3586。
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  9. Ishii,S.,Ksoll,WB,Hicks,RE和Sadowsky,MJ(2006)。&nbsp; 来自Lake Superior流域的温带土壤中归化的大肠杆菌的存在和生长。 Appl Environ Microbiol 72(1):612 -621。
  10. Ishii,S。和Sadowsky,MJ(2008)。&nbsp; 环境中的大肠杆菌:对水质和人体健康的影响。 Microbes Environ 23(2):101-108。
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引用:Somorin, Y. and O'Byrne, C. P. (2017). Determination of Survival of Wildtype and Mutant Escherichia coli in Soil. Bio-protocol 7(14): e2414. DOI: 10.21769/BioProtoc.2414.
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